NUCLEOPROTEIN STRUCTURES AND GENOME DUPLICATION

核蛋白结构和基因组复制

基本信息

批准号：
6525606
负责人：
MARCIN S FILUTOWICZ
金额：
$ 28万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-03-01 至 2005-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6525606
关键词：
DNA binding protein DNA footprinting DNA replication chimeric proteins crosslink gel mobility shift assay gene expression genetic promoter element genetic transcription methylation nucleoproteins protein sequence protein structure function reporter genes

项目摘要

DESCRIPTION (adapted from investigator's abstract): Recognition of the replication origin by initiator protein is the central event regulating DNA replication in diverse biological systems. The long term objective of this research is to understand the initiation process using the gamma origin replicon of the antibiotic resistance plasmid R6K as a model system. The R6K-encoded pi protein regulates replication and autoregulates its own expression by binding to DNA sites containing a TGARG sequence motif arranged either into seven 22-bp direct repeats (DRs) in the gamma ori, or into inverted half-repeats in the operator of its own gene pir. Pi protein has a modular structure; the C-terminus contains the DNA binding domain while the N-terminus controls pi oligomerization. The model of pi protein activity proposes that its distinct surfaces facilitate assemblies on DRs and IRs. Experiments described in Specific Aim #1 test this model using available variants of pi protein that differ from wild type pi in the control of replication and transcription. DNA and protein footprinting, photo-crosslinking and genetic fusion techniques will be employed in these investigations. In Specific Aim #2 the investigators will identify the nucleotides in pi binding sites and the amino acids in pi protein that intimately contact one another by using missing nucleotide technology and by genetically altering the DNA binding properties of pi. The investigators have recently identified a novel, non-TGAGRG pi binding site in the A+T-rich segment of gamma ori and have mapped the start of DNA synthesis approximately 30-bp upstream from this site. Specific Aim #3 will define the relationship between pi binding to this novel site and initiation by using an available in vitro replication system. The priming reaction will also be reconstituted with purified components and the effect of pi will be examined in this system. The proposed studies will reveal at a molecular level the mechanisms by which a single regulatory protein independently regulates replication and transcription.

描述（根据调查员的摘要改编）：认识引发蛋白的复制起源是调节DNA的中心事件在不同的生物系统中复制。这个长期目标研究是要使用伽马来理解启动过程抗生素抗性质粒R6K的复制子作为模型系统。这 R6K编码的PI蛋白调节复制并自动调节其自身通过结合包含布置的tgarg序列基序的DNA位点的表达在伽玛ori中分为七个22 bp的直接重复（DR），或倒入倒置在其自己的基因PIR的操作员中进行半重复。 PI蛋白具有模块化结构; C末端包含DNA结合域，而N末端控制PI低聚。 PI蛋白活性的模型提出不同的表面促进了DR和IRS上的组件。描述的实验在特定的目标＃1中，使用可用的PI蛋白变体测试该模型在控制复制和转录方面，与野生型PI不同。脱氧核糖核酸以及蛋白质足迹，照片跨链接和遗传融合技术这些调查被雇用。在特定的目标＃2中，调查人员将鉴定PI结合位点中的核苷酸和PI蛋白中的氨基酸通过使用缺失的核苷酸技术和通过基因改变Pi的DNA结合特性。研究人员最近确定了一个新颖的非TGAGRG PI结合位点在伽玛ori的A+T段中，已映射了DNA合成的开始该站点上游约30 bp。特定目标＃3将定义 PI与这个新颖站点结合与启动之间的关系可用体外复制系统。启动反应也将是用纯化的组件重构，PI的效果将在这个系统。提出的研究将在分子水平上揭示单个调节蛋白独立调节的机制复制和转录。